This invention relates to regulation of tumor suppression.
Tumors form in part as a result of disturbances in the control of cell proliferation. Alteration of one or more events in the cell cycle, particularly relating to cell division and cell differentiation, can lead to loss of control of cell multiplication. Acquisition of tumorigenicity can result from genetic changes that affect such events in the cell cycle. Generally, genetic changes such as point mutations and deletions are expected to result in a loss of function, and a genetic change that leads to tumorigenicity is likely to occur in a gene that plays a role in restraining cell multiplication. Such genes are here referred to as "tumor suppressor" genes.
SV40 large T antigen ("T") can both establish and maintain a neoplastic phenotype in responsive cell populations, and can initiate a series of events that lead to tumor formation in a suitable host. Genetic analysis has revealed a correlation between specific elements of T primary structure and transforming activity. Transformation of primary and established cells by T is dependent on a short stretch of sequence extending from residues about 105 to about 114, and a number of T mutants mapping to the 105 to 114 region have been shown to be defective in transformation.
The 105 to 114 region of T bears primary sequence and predicted secondary structure homology to one of the two transformation controlling domains of the adenovirus E1A protein domain 2, and T 101 to 108 has been shown to substitute functionally for E1A domain 2.
The product "RB" of the retinoblastoma susceptibility gene RB forms a specific complex with E1A, with T, and with E7 encoded by transforming strains of human papilloma virus. RB is a known growth regulating molecule. T-RB complex formation depends on the intact nature of the 105-114 transforming controlling region. E1A-RB complex formation depends primarily on E1A domain 2. The genetics of T-RB binding and E1A-RB binding suggest that T and E1A perform their transforming functions, at least in part, by modulating the growth regulating function of RB.
Analyses of the RB structural elements which control the T or E1A binding activity of the RB protein reveal the existence of a colinear domain of about 400 residues which alone can bind both T and E1A. Moreover, this region can specifically bind to peptide replicas of the T 105-114 and E1A domain 2 sequences and not to suitable mutant derivatives of T 105-114 or E1A domain 2. This domain of the RB protein has been termed its "pocket", because it acts as a receptacle for two viral proteins which enter it with apparently high affinity. Moreover, the RB pocket is a site for mutations resulting in spontaneous loss of function in RB, resulting in derivatives which are functionally defective in vivo and unable to bind to T or E1A in vitro. These findings suggested that the "pocket" operated, at least in part, by binding one or more cellular proteins and, therefore, that RB function was, in part, to bind to and functionally modulate certain cellular proteins with which T and E1 A competed for RB binding.
A set of such cellular proteins has recently been identified in an in vitro binding assay, and all bind only when the T binding function and the E1A binding function of the "pocket" is intact. These data support the hypothesis that at least part of the function of RB is to bind certain cellular proteins.
Domain 2 of E1A and the 105-114 region of T have been shown to form a specific complex with a second cellular protein, p107, and T binding both to RB and to p107 has been shown to be competed by a synthetic peptide spanning the 105 to 114 region in T.